Dynamic regulation of chromatin during mammalian development allows the specification of over 200 different cell types from a single genome. In addition to post-translational modification of histone proteins, selective incorporation of histone variants into chromatin adds to the complexity of epigenetic regulation. For example, histone variant H3.3 differs from canonical H3 by only 4-5 amino acids, yet displays distinct genomic and developmental enrichment properties. While long associated with gene activation, recent studies suggest that H3.3 has multiple functions in distinct genomic regions that are not always correlated with an active chromatin state. The identification of mutations in H3.3 and associated proteins in human cancers, including pediatric glioblastomas and pancreatic neuroendocrine tumors, has heightened the pressing need to understand the role of this histone variant in a developmental context. Despite increased interest, how H3.3 contributes uniquely to the function of chromatin is a long-standing, unanswered question in the field. We propose an innovative program combining mouse genetics, biochemistry, chemical biology, and cutting-edge genomics to understand the functional relevance of H3.3-mediated chromatin dynamics. Our central hypothesis is that H3.3 deposition promotes the establishment of unique developmentally regulated chromatin landscapes that allow appropriate cell fate decisions to be made. Our preliminary data suggests that nucleosome turnover rates are reduced in the absence of H3.3, and that H3.3 dynamics may facilitate histone post-translational modification states at specific genomic regions. Over the course of our studies we will (1) define the mechanisms underlying H3.3 function in promoting specific histone post-translational modification states, (2) identify regulatory mechanisms that control deposition of H3.3 at specific genomic loci, and the distribution of the histone variant between the two known chaperone systems, and (3) determine the importance of chromatin dynamics in the ability of lineage-specific transcription factors to bind target regulatory elements during cell fate transitions in the early embryo. Our proposed research is significant because it will establish chromatin variants as necessary components of cell fate transitions, and serve as a platform to understand epigenetic regulation of cell identity in both homeostasis, but also developmental misregulation and disease states.